Hydrogen peroxide initiates oxidative stress and proteomic alterations in meningothelial cells

Meningothelial cells (MECs) are fundamental cells of the sheaths covering the brain and optic nerve, where they build a brain/optic nerve-cerebral spinal fluid (CSF) barrier that prevents the free flow of CSF from the subarachnoid space, but their exact roles and underlying mechanisms remain unclear. Our attempt here was to investigate the influence elicited by hydrogen peroxide (H2O2) on functional changes of MECs. Our study showed that cell viability of MECs was inhibited after cells were exposed to oxidative agents. Cells subjected to H2O2 at the concentration of 150 µM for 24 h and 48 h exhibited an elevation of reactive oxygen species (ROS) activity, decrease of total antioxidant capacity (T-AOC) level and reduced mitochondrial membrane potential (ΔΨm) compared with control cells. 95 protein spots with more than twofold difference were detected in two dimensional electrophoresis (2DE) gels through proteomics assay following H2O2 exposure for 48 h, 10 proteins were identified through TOF/MS analysis. Among the proteomic changes explored, 8 proteins related to energy metabolism, mitochondrial function, structural regulation, and cell cycle control were downregulated. Our study provides key insights that enhance our understanding of the role of MECs in the pathology of brain and optic nerve disorders.

Optic nerve and brain are enveloped with three meninges including dural, arachnoid and pial sheaths. Meningothelial cells (MECs) are predominant cell components covering both the arachnoid and the pia mater of the neuronal tissue and closely contact with cerebral spinal fluid (CSF), which facilitates to build a brain/optic nerve-CSF barrier that prevents the free flow of CSF from the subarachnoid space. MECs are crucial for removal of the active biomolecules through phagocytosis from CSF to maintain the micro-environment balance of the subarachnoid space. Therefore, any pathophysiological changes in MECs might have an impact on the integrity of the brain/optic nerve-CSF barrier [1][2][3][4][5] .
Increasing investigations have demonstrated that oxidative stress is involved in the progression of numerous neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, glaucoma, and mitochondrial optic neuropathies [6][7][8][9] . The brain and optic nerve are particularly sensitive to oxidative damage due to their high metabolic rate but with a limited capacity of cellular regeneration 9 . Oxidative stress characterized by the overproduction of free radicals plays a pivotal role in these neurodegenerative diseases. Mitochondria have been recognized to be a major site for reactive oxygen species (ROS) production 10 . ROS serve as important signaling molecules, whereas the over-accumulation of ROS in pathological conditions results in oxidative stress 11 . Mitochondrial membrane potential (ΔΨm) is crucial for sustaining the biological activities of the respiratory chain to generate adenosine triphosphate (ATP) and maintaining the normal function of mitochondria, the loss of ΔΨm deprives cells of energy and leads to subsequent death 12,13 . Brain and ocular tissues normally have the potential to balance the mild damage caused by oxidative stress through several intrinsic antioxidant enzymes. However, overproduction of ROS, free radicals, and mitochondrial dysfunction overwhelms the intrinsic antioxidant capacity and results in oxidative stress and progression of pathological damages 7 . We speculate that oxidative stress leads to over-accumulation of ROS, mitochondrial dysfunction in MECs, thereby impairing the protective role of MECs in maintaining the integrity of the brain/optic nerve-CSF barrier, which probably contributes to the brain and optic nerve disorders.
The present study was undertaken to determine the influence of hydrogen peroxide (H 2 O 2 ) induced-oxidative stress on cellular functional changes of MECs. Proteomics approach was conducted to detect the protein expression alteration profile upon the oxidative stress. We expected that the results of this study provides new insights into the further understanding of the roles of MECs.

Exposure to H 2 O 2 inhibits cell viability of MECs. To investigate the effects of H 2 O 2 on cell viability of
MECs, cells were exposed to various concentrations of H 2 O 2 ranging from 25 to 250 μM for 12 h, 24 h, 36 h, and 48 h respectively. Cell viability in H 2 O 2 -exposed cell groups obviously decreased compared with control cells, which showed an inhibition effect of H 2 O 2 on cell growth. Significant inhibition effect was presented when cells were incubated with H 2 O 2 at a concentration of 150 μM at the time of 12 h (p < 0.001) (Fig. 1A). Furthermore, a more potent suppressive effect on the MECs cell viability was exhibited with the increase of both the H 2 O 2 exposure time and treatment concentration ( Fig. 1B-D). H 2 O 2 alters the morphology of MECs. To identify cellular morphological changes upon the oxidative stress, we observed cells under the inverted microscope. We found that MECs from controls showed increased cellular proliferation and round nuclear after the incubation for 24 h and 48 h ( Fig. 2A,C). In contrast, the proliferation of cells treated with H 2 O 2 at a concentration of 150 μM for the same period was inhibited (Fig. 2B,D). The MECs configuration made a change from round shape into shrunk and elongated ones in response to H 2 O 2 compared to the control group (Fig. 2B www.nature.com/scientificreports/ applied for detecting mitochondrial depolarization. MECs were harvested and processed for JC-1 detection with flow cytometry after different treatment. Our results showed that J-aggregates were decreased in H 2 O 2 -exposed cells when compared to the controls (Fig. 5A,B). There was a significant increase in monomer in cell groups with  www.nature.com/scientificreports/ gels revealed that 95 protein spots were differentially expressed in the two cell groups. Compared to the nontreated cells, 15 proteins were upregulated and 80 were consistently downregulated with fold change ≥ 2-folds (p < 0.05). The protein spots appeared in the 2-DE gel images of the control (Fig. 6A) and H 2 O 2 -treated MECs (Fig. 6B). Proteins with masses varying between 20 and 117 kDa were separated in large format gels along a pH interval of 3-10. In order to obtain the highly significant differentially expressed proteins from our study, the protein differential expression was defined by the criteria of fold change ≥ 3-folds (p < 0.01), and a total of 10 significant differentially expressed proteins between the controls and the H 2 O 2 -treated cells were identified. Among these 10 proteins, 8 proteins were downregulated and 2 proteins were upregulated. Table 1 showed Swiss-Prot Accession Numbers, full protein names, theoretical molecular weight (MW), isoelectric point (PI) as well as protein coverage of the identified proteins.

Discussion
Emerging evidence demonstrated that MECs is highly active to pathological stress, such as involving in the activation of the immune response by releasing pro-inflammatory cytokines and producing lipocalin-type prostaglandin D synthase to modulate CSF composition under pathological conditions [14][15][16] . MECs also have a potential  www.nature.com/scientificreports/ role for phagocytosis to remove active molecules or waste products from the subarachnoid space to keep the integrity of brain/ optic nerve-CSF barrier 4 . Impairment of MECs function may be a contributing factor to the disorders of brain and optic nerve. H 2 O 2 is a key oxidant to induce free radical and apoptosis, and is therefore widely used in experimental studies as an oxidative stress. H 2 O 2 has multiple biological effects including mediation of cell proliferation, migration,   In this study, proteomics approach was performed to explore the protein expression profile of MECs to identify proteins involved in the pathogenesis of oxidative stress. A total of 10 stably, significantly dysregulated proteins were detected. Among these identified proteins, vigilin, as a highly conserved protein from yeast to mammals, was downregulated in our study. The diversity of vigilin's biological roles includes chromosome segregation, translation and tRNA transport, and regulation of mRNA metabolism 22,23 . In our study, down-regulation of vigilin in H 2 O 2 -treated cells might contribute to the inhibition of cellular function to regulate the translational activities of mRNAs under the oxidative stress.
Three actin regulatory-related proteins which were downregulated in our present study include vimentin, macrophage-capping protein (CAPG), and Cofilin-1. Vimentin performs a significant role in supporting and anchoring the position of the cellular organelles by attaching to the nucleus, endoplasmic reticulum and mitochondria. It is also the cytoskeletal component responsible for maintaining cell integrity, cell adhesion and extracellular matrix (ECM) formation 24,25 . The lack of vimentin results in the loss of cell morphology and reduces cell adhesion 26 . In addition, vimentin plays a role in regulating the membrane potential of mitochondria and supporting its high bioenergetic capacity, and vimentin deficiency inhibits ATP synthesis and promotes ROS production under pathological conditions 25,26 . It is highly likely that the decreased vimentin expression upon the oxidative stress in our study may influence its function of maintaining cell shape, integrity of the cytoplasm, stabilizing cytoskeletal interactions and mitochondrial function. CAPG has been identified to be an actin-binding protein and displays a range of activities including regulating cytoplasmic and nuclear structures, modulating cell motility by interacting with the cytoskeleton 27,28 . Cofilin-1 is a nonmuscle isoform of actin regulatory protein that belongs to the cofilin family 29 . Studies of the cofilin family have demonstrated that this molecule regulates a complex series of events such as actin filament turnover, cell cycle progression, migration, cell motility, and formation of cell processes [30][31][32] . In our study, the dysregulation of CAPG, vimentin, and Cofilin 1 in MECs upon H 2 O 2 stress could be an important contributing factor to the cell viability loss, cell morphology alteration, and mitochondrial dysfucntion of MECs.
Our study showed that both ubiquitin and nucleoside diphosphate kinases (NDPKs) levels were suppressed in cells subjected to oxidative stress. Ubiquitin was recognized to be a crucial molecule to label intracellular proteins for degradation by a multienzymatic complex 33,34 . E2 enzyme for interferon-stimulated gene-15 (ISG15) is responsible for the attachment of ubiquitin (Ub) to cellular proteins 35 . NDPKs are key enzymes ubiquitously found in all organisms and show remarkable sequence conservation 36 . They catalyze the transfer of terminal phosphates from nucleoside triphosphates (NTP) to nucleoside diphosphates (NDP) to yield their respective www.nature.com/scientificreports/ nucleoside triphosphates 37 . NDPKB is a DNA-binding protein that recognizes the nuclease-hypersensitive element and maintains the intracellular concentration of NTPs and dNTPs. The downregulation of enzymes including Ubiquitin/ISG15-conjugating enzyme E2 and NDPKB in our study suggests that oxidative stress most likely attenuates their enzymatic role in multiple cellular processes such as cell proliferation, differentiation, and cell development.
Our protemic analysis demonstrated that H 2 O 2 inhibited the expression of cytoplasmic dynein intermediate chain 2, which is an essential component of the dynein complex and exerts important functions in its cargo recognition, assembly, and regulation [38][39][40] . The down-regulation of cytoplasmic dynein intermediate chain 2 in H 2 O 2 -exposed MECs indicates a possible mechanism by which the oxidative stress inhibits dynein-dependent transport, alters dynein motility, affects its major roles in communicating with other protein complexes 41 , and thereby leading to the consequent changes in MECs morphology, adhesion and migration. Dynein involves in the subcellular distribution of vimentin intermediate filaments that are crucial cytoskeletal components contributing to cell shape, motility and organelle positioning 42,43 . The dysfunction of these proteins is consistent with our microscopic observation that MECs cell shape is altered with the proteomic changes. Mitochondrial stress-70 protein is one of heat shock proteins and the most important chaperone found in the mitochondrial matrix 44 . Mitochondrial stress-70 protein plays a crucial role in import and refolding of mitochondrial proteins and is essential for protecting intracellular proteins from heat shock, toxicity, hypoxia and inflammation, ROS accumulation and mitochondrial dysfunction [45][46][47][48] . In our experiments, decreased expression of stress-70 protein in the stressed cells may be linked to the increased vulnerability of MECs to the oxidative stress.
Data from our study showed that H 2 O 2 upregulated guanine nucleotide-binding protein subunit beta (GNBP-B) expression. GNBP-B is involved in several crucial biological processes including modulating various transmembrane signaling systems for cell growth, cellular response to hypoxia, and cell apoptosis 49 . Besides GNBP-B, our proteomic data revealed that ATP synthase subunit alpha was also elevated in cells exposure to H 2 O 2 . Mitochondrial membrane ATP synthase produces ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain 50,51 . Upregulation of GNBP-B and ATP synthase subunit alpha in our study implies that elevated level of these proteins is associated with an increased demand for energy production by the dysfunctional cells to maintain basic function under the oxidative stress.
MECs, the predominant cellular population at the interface between CSF and neuronal tissue, play a pivotal role in maintaining the function of brain/optic nerve-CSF barrier. In our study, H 2 O 2 -initiated stress decreased cell viability, altered cell morphology, increased intracellular ROS activity and reduced T-AOC level and ΔΨm, indicating that MECs are susceptible to the oxidative-stress attack. Among the proteomic changes explored, proteins related to energy metabolism, mitochondrial function, structural regulation, and cell cycle control were dysregulated in response to H 2 O 2 -triggered stress, which may consequently affect cell viability, oxidant-antioxidant balance, and mitochondrial function of MECs, a possible bio-toxic effect due to the malfunction of brain/optic nerve-CSF barrier and reduced clearance of highly biological active molecules in CSF may ensue. Therefore, any impairment of MECs function probably disturbs the integrity of the brain/optic nerve-CSF barrier, which may involve in the disorder of micro-environment of the subarachnoid space surrounding the brain and optic nerve.
In conclusion, this work provides new insights into the cellular mechanisms associated with the pathophysiology of MECs subject to oxidative stress. These findings may be used as a basis for further understanding the role of MECs in brain and optic nerve disorders.

Materials and methods
Cell culture. Human meningothelial cell line (Ben Men cell line) (DSMZ, Germany) was cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/mL, 100 µg/mL; Sigma, Germany). Cells were trypsinized after washed with phosphate-buffered saline (PBS) (Sigma, Germany), and supernatant was removed after centrifugation.  Gel scan and analysis. The gels were run in triplicate for each sample and stained with silver nitrate solution. The stained gel was scanned by the Image Scanner (GE Healthcare, USA) at a resolution of 300 dots per inch. All gel images were processed by three steps: spot detection, volumetric quantification, and matching, using PDquest 8.0 software.

Digestion.
For gel digestion, the gel spot was destained at room temperature for 5 min after the gel spot was washed twice, removed and incubated in 50% acetonitrile (ACN) and 100% ACN. The gels were rehydrated in 2-4 μL trypsin (Promega, Madison, USA) solution for 30 min. Cover solution (25 mmol/L NH 4 HCO 3 ) was then added for digestion for 16 h at 37 °C. The supernatants were transferred into another tube, and the gels were extracted once with 50 μL extraction buffer [67% ACN and 5% trifluoroacetic acid (TFA)]. The peptide extracts and the supernatant of the gel spot were combined and then completely dried.

MS analysis.
Samples were re-suspended with 5 μL 0.1% TFA followed by mixing in 1:1 ratio with a matrix consisting of a saturated solution of α-cyano-4-hydroxy-trans-cinnamic acid in 50% ACN, 0.1% TFA. Mixture (1 ul) was spotted on a stainless steel sample target plate. Peptide MS and MS/MS were performed on an ABI 5800 MALDI-TOF/TOF Plus mass spectrometer (Applied Biosystems, Foster City, USA). Data were acquired in a positive MS reflector using a CalMix5 standard to calibrate the instrument (ABI5800 Calibration Mixture).
Statistical analysis. Independent t-test or ANOVA followed by Bonferroni's post hoc test was applied.
Data were expressed as the mean ± standard deviation. For all statistical analyses, the level of significance was set at a probability of 0.05. Statistical analyses were conducted with SPSS 19.0 statistical analysis software (SPSS Inc., Chicago, IL).